PROJECT SUMMARY Schizophrenia (SZ) results from the complex interplay of genetic variants affecting liability and environmental risk factors that alter developmental trajectories of neural circuits. Despite this etiological diversity, alterations in GABA neurons are a highly conserved feature of SZ and substantial data suggest these alterations contribute to cognitive dysfunction in the disorder. GABA neurotransmission is heavily dependent on GAD67. Many GABA neurons in the PFC of SZ subjects express normal levels of GAD67 mRNA, but this transcript is not detectable in 30-50% of PFC GABA neurons in the disorder. The identities of the affected GABA neurons are largely unknown. Transcriptionally-unique subtypes (TUS) of GABA neurons uniquely target distinct pyramidal neuron (PN) ensembles, allowing them to powerfully modulate certain brain-wide networks and behaviors. Thus, knowing the identity of the affected GABA TUS in SZ and the mechanisms underlying GAD67 mRNA deficits occurring in only some GABA TUS is essential for moving towards a mechanistic understanding of the cortical circuitry alterations in the illness. To meet these needs, the following Aims are proposed: Aim 1. Quantify GAD67 mRNA and protein levels in the soma and axon terminals, respectively, of GABA TUS in SZ. Information from profiling studies of GABA neurons in mice, was used to identify mRNA markers that distinguish 10 GABA TUS in human PFC, which capture >95% of all GABA neurons. Multiplex fluorescence in situ hybridization (FISH) is used to quantify GAD67 mRNA levels within these TUS in the PFC of matched SZ and control (CON) subjects (Cohort 1). In the same subjects, multi-label immunohistochemistry is used to quantify GAD67 protein levels in the axon terminals of the different GABA TUS. The GAD67 mRNA deficit in SZ is predict to be restricted to specific GABA TUS, which have lower terminal GAD67 protein levels. Aim 2. Determine the pattern of altered TUS that is specific to the disease process of SZ. Some alterations in cortical GABA neurons, including lower levels of GAD67, seem to be shared across SZ, bipolar disorder (BP), and major depressive disorder (MDD), whereas those of others are not. The studies of Aim 1 are conducted in an all new second cohort of matched SZ, BP, MDD and CON subjects. SZ, BP, and MDD are predicted to each have a unique constellation of affected GABA neuron subtypes relative to each other. Aim 3. Perform transcriptome sequencing of GABA TUS in SZ and CON subjects. In Cohort 1 subjects, individual neurons of each GABA TUS are identified by FISH, collected using laser microdissection, pooled and subjected to transcriptome sequencing. In SZ, the affected GABA TUS are predicted to share a pattern of differentially-expressed genes that are components of pathways known to regulate GAD67 expression. By identifying the SZ-specific constellation of affected GABA TUS and assessing their transcriptome profile, these studies will provide essential information for future mechanistic tests of TUS-specific GABA neuron circuit dysfunction in model systems and developing therapeutic targets of GABA TUS.